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Glycerol channel protein gene deleted brewing microzyme strain capable of reducing glycerol output and increasing ethanol output and construction method thereof

A technology of Saccharomyces cerevisiae strain and channel protein, which is applied in the field of Saccharomyces cerevisiae biological fermentation, can solve the problems of long fermentation period, environmental pollution, and high fermentation by-products, reduce the production of glycerol by-products, solve high production costs, and increase ethanol production Effect

Active Publication Date: 2006-04-26
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there are mainly the following series of problems in the production of ethanol by industrial fermentation at home and abroad: high fermentation by-products, long fermentation cycle, low ethanol conversion rate and serious environmental pollution and other major problems
[0003] Therefore, genetic engineering was used to delete the gene encoding the glycerol channel protein FPS1 embedded in the cell membrane of Saccharomyces cerevisiae

Method used

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  • Glycerol channel protein gene deleted brewing microzyme strain capable of reducing glycerol output and increasing ethanol output and construction method thereof
  • Glycerol channel protein gene deleted brewing microzyme strain capable of reducing glycerol output and increasing ethanol output and construction method thereof
  • Glycerol channel protein gene deleted brewing microzyme strain capable of reducing glycerol output and increasing ethanol output and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] (1) Acquisition of haploid Saccharomyces cerevisiae and deletion of URA3 gene

[0038] Saccharomyces cerevisiae (S. cerevisiae) can exist in nature both in haploid and diploid form. Under normal circumstances, it will sprout and multiply in the state of vegetative body; the vegetative body can be haploid (n) It can exist as a diploid (2n); it can only reproduce sexually under certain environmental conditions. One ascus produces four ascus buns, two of which are type a and two are type α. .

[0039] The formation of ascospores in Saccharomyces cerevisiae: Take appropriate amount of newly activated diploid S. cerevisiae cells and spread them evenly on the ascospore-producing medium with potassium acetate, and culture them at 28°C for 2 days. Scrape an appropriate amount of yeast cells from the coating, dissolve them in a 1.5ml centrifuge tube filled with 50μl sterile water, add 5μl zymolyase at a concentration of 15mg / ml to digest the ascos wall at 37°C for 20min to form Sacc...

Embodiment 2

[0068] (1) Acquisition of haploid Saccharomyces cerevisiae and deletion of URA3 gene

[0069] Saccharomyces cerevisiae ascospore formation: Take appropriate amount of newly activated diploid Saccharomyces cerevisiae (S. cerevisiae) cells uniformly spread on potassium acetate ascospore production medium, culture at 26 ℃ for 3 days, in the medium yeast Scrape an appropriate amount of yeast cells from the coating, dissolve them in a 1.5ml centrifuge tube containing 50μl of sterile water, add 10μl of zymolyase at a concentration of 10mg / ml to digest the ascosal wall at 37°C for 10min to form Saccharomyces cerevisiae ascospores Solution

[0070] Saccharomyces cerevisiae ascospore resolution: slowly add 1ml of sterile water to the centrifuge tube containing the ascospore solution of Saccharomyces cerevisiae, invert for 4min, take 50μl of the liquid, drop it on the edge of the YPD plate, tilt the plate to make the cells The liquid forms a strip on the edge of the plate. After drying, the...

Embodiment 3

[0083] (1) Acquisition of haploid Saccharomyces cerevisiae and deletion of URA3 gene

[0084] Saccharomyces cerevisiae ascospore formation: Take appropriate amount of newly activated diploid Saccharomyces cerevisiae (S. cerevisiae) cells uniformly spread on potassium acetate ascospore production medium, culture at 30 ℃ for 2 days, in the medium yeast Scrape an appropriate amount of yeast cells from the coating, dissolve them in a 1.5ml centrifuge tube containing 50μl of sterile water, add 3μl of zymolyase at a concentration of 20mg / ml to digest the ascus wall at 37°C for 30min to form Saccharomyces cerevisiae ascospores Solution

[0085] Resolution of ascospores of Saccharomyces cerevisiae: the steps are the same as in Example 2;

[0086] Deletion of URA3 gene of KAM-19 strain: the same steps as in Example 1;

[0087] (2) Construction of PUC18-FPS1-ORF plasmid

[0088] Preparation of yeast chromosomes: the steps are the same as in Example 2;

[0089] Amplify the FPS1-ORF gene: the...

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Abstract

The present invention discloses one kind of Saccharomyces cerevisiae strain with glycerin passage protein gene deficiency and resulting in lowered glycerin generation and raised ethanol yield and its construction process. The construction process includes the following steps: 1. obtaining Saccharomyces cerevisiae monoploid and deleting of URA3 gene; 2. constructing PUC18-FPS1-ORF plasmid; 3. constructing PUC18-FUF plasmid; and 4. constructing KAM-21 strain. The present invention deletes glycerin passage protein FPS1 gene code embedded on cell membrane and loses the expression of FPS1 gene by means of gene engineering technology, so as to inhibit glycerin synthesizing way, convert from glycerin metabolism to ethanol metabolism, increase ethanol yield, and solve serial problems in ethanol production.

Description

Technical field [0001] The invention belongs to the field of Saccharomyces cerevisiae biological fermentation, specifically the ethanol fermentation industry. In particular, it relates to a strain of Saccharomyces cerevisiae modified by genetic engineering and a method for constructing the strain. Background technique [0002] Ethanol has been a renewable energy source that partially or completely replaces petroleum, and is of great significance to the sustainable development of human society. However, the main problems in the production of ethanol by industrial fermentation at home and abroad are as follows: high fermentation by-products, long fermentation cycle, low ethanol conversion rate and serious environmental pollution. Glycerol is the main by-product in the ethanol fermentation process, which consumes about 10% of fermentable sugars. The production of glycerol by-products directly affects the sugar-alcohol conversion rate. It has become a major obstacle to reducing the c...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/09C12R1/865
Inventor 马平生张爱利孔庆学赵学明
Owner TIANJIN UNIV
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